There is evidence that the necrotic lesions resulting from spider bites are due to the spider's digestive protease which contaminate the venom during such encounters. It has been hypothesized that collagenolytic proteases are responsible. The only site-specific inhibitors of spider proteases known at the time are those occurring in the spider's hemolymph (blood). There is no established course of treatment for the spreading necrosis following spider bites at present. Should the foregoing hypothesis be correct, and increase in our knowledge of the spider proteases and their inhibitors should allow for the development of a more effective treatment for necrotic arachnidism. We propose to isolate two collagenolytic proteases from the digestive fluid and one (or possibly two) protease inhibitor(s) from the hemolymph of the spider Argiope aurantia. The collagenolytic proteases will be characterized according to molecular weight, subunit composition, amino acid composition, partial amino acid sequence, metal ion content, and cleavage specificity to establish their mechanistic class. The isolated protease inhibitor(s) will be characterized according to molecular weight, subunit composition, and partial amino acid sequence. We will explore the effectiveness of the inhibitor(s) against the various classes of proteases to establish its/their range of inhibitory activity. We will use the purified spider proteases and their inhibitor(s) in laboratory trials on mice to examine the hypothesis that spider digestive proteases are the cause of spider bite tissue necrosis. Pending the outcome of the proposed study, future studies will examine the use of the Argiope protease inhibitor(s) in the treatment of the bites of those spiders producing the most severe lesions(brown recluse and hobo spiders). Through oligonucleotide probe synthesis and genomic DNA library screening, we will use the partial amino acid sequences from the present study to establish the entire amino acid sequences of the proteases and inhibitor(s) and make a database search for homologous sequences. We will continue our exploration of the potential of the spider proteases in biomedical research.